Exonuclease enhances hybridization efficiency: improved direct cycle sequencing and point mutation detection.
Solution hybridization is an essential step in sequencing and some point mutation detection methods. In practice, this hybridization is hampered resulting in the need of additional purification of the amplification products. The use of T7 gene 6 exonuclease may lead to efficient production of single-stranded DNA. In this study, the effect of pretreatment with exonuclease on direct cycle sequencing and point mutation detection was analyzed. Exonuclease-treated products were directly cycle sequenced without further purification. This resulted in highly efficient quality improvement for sequencing allowing detection of heterozygotes. Point mutation detection by Point-EXACCT (exonuclease-amplification coupled capture technique) demonstrated detection of one cell containing a mutation in an excess of 75000 wild type cells. Exonuclease-enhanced detection methods offer simple, rapid detection strategies that are easily adaptable for widespread clinical laboratory use. With the use of exonuclease, the detection of heterozygosity using fluorescent cycle sequencing is becoming more reliable. The high sensitivity of Point-EXACCT due to the use of exonuclease makes it a highly promising method for large-scale screening of (pre)malignant changes in patients with a high risk for developing cancer.
Somers
V A
VA
Department of Pathology, Maastricht University, The Netherlands. vso@lpat.azm.nl
Leimbach
D A
DA
Murtagh
J J
JJ
Thunnissen
F B
FB
D004252:DNA Mutational Analysis; D015342:DNA Probes / Q000737:chemistry; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005453:Fluorescence; D011905:Genes, ras / Q000235:genetics; D005820:Genetic Testing; D006579:Heterozygote; D006801:Humans; D009369:Neoplasms / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D017354:Point Mutation / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
D015342:DNA Probes; D005090:Exodeoxyribonucleases; C046264:gene b exonuclease